Colony-stimulating factor 3 receptor (CSF3R) M696T mutation does not impact on clinical outcomes of a Ph+ acute lymphoblastic leukemia patient

Abstract

Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in a variety of myeloid disorders. Although CSF3R point mutations (eg, T618I) are emerging as key players in chronic neutrophilic leukemia/atypical chronic myelogenous leukemia , the significance of rarer CSF3R mutations is unknown. Here, we report a 32-year-old female who was diagnosed as Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph⁺ ALL) with the CSF3R M696T mutation and was undergone unrelated donor hematopoietic stem cell transplantation. The patient achieved complete remission with chemotherapy in combination with tyrosine kinase inhibitor (TKI) and long-term survival by unrelated donor transplantation. Meanwhile, we performed a series of experiments using murine interleukin 3 (IL-3)-dependent Ba/F3 cell line to evaluate the transforming capacity of the CSF3R M696T mutation. We confirmed the presence of a CSF3R M696T germline mutation in this patient which was inherited from her mother. The in vitro experiment results showed that the CSF3R M696T mutation contributes marginally to the tumor transformation of Ba/F3 cells, indicating that CSF3R M696T mutation was neutral in tumor transformation ability. We concluded that TKI is effective in patients with the CSF3R M696T mutation in Ph⁺ ALL and donors with CSF3R M696T mutation might still be selected as the candidate for transplantation.

Keywords: CSF3R M696T mutation; Familial inheritance; Pathogenicity; Ph+ acute lymphoblastic leukemia.

Figure 1

Clinical treatment and pedigree analysis of CSF3R gene mutation in this patient. (A) Clinical treatment of this patient. (B) Pedigree analysis of CSF3R gene mutation in this patient. BM = bone marrow morphology, CAM = cyclophosphamide + cytarabine + mercaptopurine, CR = complete response, MRD = minimal residual disease, PBSCT = peripheral blood hematopoietic stem cell transplantation, URD = unrelated donor, VP = vindesine + prednisone), VTLP = vinorelbine + pirarubicin + Pegaspargase + prednisone, VTCP = vindesine + pirarubicin + cyclophosphamide + prednisone.

Figure 2

Tumor transformation ability and G-CSF dependency for various CSF3R mutations in Ba/F3 cells. (A) Csf3r mRNA levels quantified by RT-qPCR (n = 3). WBM, whole bone marrow cells of mice; Gran, granulocytes from bone marrow cells of mice, Gr-1⁺Mac-1⁺. Both two types of cells above were positive control due to their high expression of Csf3r. Data were normalized to 18S expression. ^∗∗∗P < .001. (B and C) Relative RNA and protein expression of CSF3R in Ba/F3 cell lines expressing wild-type CSF3R and its three mutations (n = 3). Parental Ba/F3 cells and empty vector (EV) infected Ba/F3 cells were used as negative controls. ^∗∗∗P < .001. (D and E) Relative cell viabilities of transduced Ba/F3 cells were measured in the presence (D) or absence (E) of IL-3. (F) Transduced Ba/F3 cells were cultured with a concentration gradient of G-CSF following IL-3 withdrawal and assessed by CCK8 assay at 72 hours. Data were normalized to parental Ba/F3 cells without G-CSF stimulation. (G) Western blotting analysis of the phosphorylation levels and total protein levels of Stat3 and Src in Ba/F3 cells expressing CSF3R mutations after 5 days culture in the presence and absence of IL-3. Parental Ba/F3 cells and Ba/F3 cells expressing wild-type CSF3R were used as controls. β-Actin and GAPDH as the internal controls. EV = empty vector.